Spatial and temporal variations reveal the response of zooplankton to cyanobacteria

The effects of cyanobacteria on zooplankton abundance, structure and diversity were investigated, based on a systematic study on spatial and temporal variations of cyanobacteria and zooplankton in Lake Taihu from 1998 to 2007. It was found that similar increasing trends of cyanobacteria/phytoplankton ratios were accompanied by different trends in biomass, composition and biodiversity of zooplankton in different regions of the lake; the cladocerans benefitted from the increase in cyanobacteria; however, rotifers and protozoans were negatively affected by cyanobacteria. The biomass-based biodiversity of phytoplankton and zooplankton was negatively affected by cyanobacteria as well, and the adverse effects were in proportion to the cyanobacteria/phytoplankton ratio. These results indicated interestingly that higher amounts of cyanobacteria do not necessarily reduce zooplankton biomass, as the biomass of larger zooplankton such as cladocerans was positively related to cyanobacteria. The findings are essential to understand the complex ecological effects of cyanobacteria blooms in lakes.     

Phylogenetic analysis and molecular signatures defining a monophyletic clade of heterocystous cyanobacteria and identifying its closest relatives

Detailed phylogenetic and comparative genomic analyses are reported on 140 genome sequenced cyanobacteria with the main focus on the heterocyst-differentiating cyanobacteria. In a phylogenetic tree for cyanobacteria based upon concatenated sequences for 32 conserved proteins, the available cyanobacteria formed 8–9 strongly supported clades at the highest level, which may correspond to the higher taxonomic clades of this phylum. One of these clades contained all heterocystous cyanobacteria; within this clade, the members exhibiting either true (Nostocales) or false (Stigonematales) branching of filaments were intermixed indicating that the division of the heterocysts-forming cyanobacteria into these two groups is not supported by phylogenetic considerations. However, in both the protein tree as well as in the 16S rRNA gene tree, the akinete-forming heterocystous cyanobacteria formed a distinct clade. Within this clade, the members which differentiate into hormogonia or those which lack this ability were also separated into distinct groups. A novel molecular signature identified in this work that is uniquely shared by the akinete-forming heterocystous cyanobacteria provides further evidence that the members of this group are specifically related and they shared a common ancestor exclusive of the other cyanobacteria. Detailed comparative analyses on protein sequences from the genomes of heterocystous cyanobacteria reported here have also identified eight conserved signature indels (CSIs) in proteins involved in a broad range of functions, and three conserved signature proteins, that are either uniquely or mainly found in all heterocysts-forming cyanobacteria, but generally not found in other cyanobacteria. These molecular markers provide novel means for the identification of heterocystous cyanobacteria, and they provide evidence of their monophyletic origin. Additionally, this work has also identified seven CSIs in other proteins which in addition to the heterocystous cyanobacteria are uniquely shared by two smaller clades of cyanobacteria, which form the successive outgroups of the clade comprising of the heterocystous cyanobacteria in the protein trees. Based upon their close relationship to the heterocystous cyanobacteria, the members of these clades are indicated to be the closest relatives of the heterocysts-forming cyanobacteria.     

T7 RNA聚合酶基因表达系统在鱼腥藻7120中构建及hG-CSF的表达

外源基因的表达效率低是蓝藻基因工程发展的瓶颈之一,T7 RNA聚合酶表达系统实现了大肠杆菌中外源基因的高效表达,蓝藻与大肠杆菌同为革兰氏阴性菌,具有较高的遗传同源性,在蓝藻中构建T7 RNA聚合酶表达系统有可能提高外源基因在蓝藻中的表达效率。为了在鱼腥藻7120中构建T7 RNA聚合酶表达系统,采用重叠延伸PCR技术和酶切连接等方法构建能够表达T7 RNA聚合酶的定点整合载体pEASY-T1-F1-TacT7RNAPCmR-F2以及由T7启动子驱动hG-CSF基因表达的穿梭表达载体pRL-T7-hG-CSF;采用电击转化法将定点整合载体导入野生型鱼腥藻中,通过三亲接合的方法将穿梭表达载体转入已定点整合T7 RNA聚合酶的转基因鱼腥藻中。利用PCR技术鉴定外源基因在蓝藻中的存在;RT-PCR方法检测外源基因在蓝藻中的转录情况;Western blotting实验检测外源基因在蓝藻中的蛋白表达情况。结果表明两种载体构建成功,T7 RNA聚合酶基因和hG-CSF基因被转入鱼腥藻中,两个基因均在藻中表达,T7 RNA聚合酶表达系统在鱼腥藻中构建成功,与传统蓝藻表达系统相比,文中在鱼腥藻中构建的T7表达系统使hG-CSF基因的表达量提高2倍。该表达系统将为蓝藻基因工程的应用提供更优的工具,将促进蓝藻作为底盘细胞在合成生物学等领域的发展。     

Automation of species-specific cyanobacteria phycocyanin fluorescence compensation using machine learning classification

High-frequency cyanobacteria monitoring often uses in-situ fluorescence of phycocyanin (f-PC). However, f-PC must be calibrated for the dominant cyanobacteria species, and it cannot distinguish cyanobacteria taxa, which relies on conventional time-consuming cyanobacteria identification methods. This study proposes a framework to automate f-PC species-specific compensation through three components: (1) prediction of the dominant cyanobacteria species using data-driven models and routine environmental monitoring data; (2) determination of species-specific f-PC per biomass in controlled laboratory experiments; and (3) automation of f-PC species compensation. The framework was validated by applying it to Myponga drinking water reservoir in South Australia. Three machine learning techniques using only high-frequency water temperature data were compared to predict the dominant cyanobacteria species. The framework application to Myponga drinking water reservoir improved the agreement of f-PC with conventional cyanobacteria biovolume measurements, and provided rapid, low-cost identification of the dominant cyanobacteria species, which can support proactive species-targeted cyanobacteria management.     

A new method for the rapid evaluation of gas vacuoles regeneration and viability of cyanobacteria by flow cytometry

Flow cytometric analysis for measuring gas vacuole regeneration and viability of cyanobacteria was developed. This novel approach distinguished between cyanobacteria with intact and collapsed gas vacuoles. By this method, sonicated cyanobacteria under illuminated conditions were shown to regenerate their gas vacuoles to the level of the untreated cells within 1–3 days. Ultrasonically treated cyanobacteria cultured under non-aerated and non-illuminated conditions did not regenerate their gas vacuoles. Combined with dual staining, viable and non-viable cyanobacteria are easily and rapidly quantified or enumerated by measuring the intensity of red fluorescence and green fluorescence. A high correlation was found between the numbers of viable and non-viable cyanobacteria with flow cytometric measurement.     

蓝藻基因转移系统的选择与建立

蓝藻是一类进行光合放氧的原核生物 ,因其结构的特殊性 ,已成为表达外源基因的理想宿主之一。然而外源基因转化系统的选择与建立一直影响着蓝藻基因工程的快速发展。总结了各类蓝藻基因转移系统的特点、影响因素、各系统间的优缺点、以及不同蓝藻株系最适基因转移系统的选择等 ,为利用蓝藻进行遗传操作提供可能 ,为蓝藻基因工程发展提供信息。     

Molecular phylogeny and evogenomics of heterocystous cyanobacteria using rbcl gene sequence data

Taxonomic affiliations and molecular diversity of 41 heterocystous cyanobacteria representing 12 genera have been assessed on an evolutionary landscape using rbcl gene sequence data-based phylogenomics and evogenomics approaches. Phylogenetic affiliations have clearly demonstrated the polyphyly of the true branching cyanobacteria, along with a frequent intermixing amongst the heterocystous cyanobacteria. The monophyletic origin of the heterocystous cyanobacteria was also quite evident from maximum parsimony and neighbor joining analyses. Incongruency with the traditional scheme of cyanobacterial taxonomy was frequently observed, thus advocating towards some re-amendments in the cyanobacterial classificatory schemes. Evogenomics analyses of gene sequence data gave a clear indication about the greater evolutionary pace of the unbranched cyanobacteria as compared to the branched forms. It was evident that the order Nostocales would be controlling the future pace of evolution of heterocystous cyanobacteria. The cyanobacteria Nostoc was found to have the greatest genetic heterogeneity amongst the studied genera, along with some evidence towards events of lateral gene transfer amongst the heterocystous cyanobacteria in case of the rbcl gene. Thus, heterocystous cyanobacteria were found to be a fast evolving group, with estimates of gene conversion tracts pointing towards the unbranched heterocystous cyanobacteria being at the base of evolutionary diversifications of the complete heterocystous lineage.     

Occurrence of sym-homospermidine as the major polyamine in nitrogen-fixing cyanobacteria

We analyzed the amount of polyamines in a variety of cyanobacteria including nitrogen-fixing and nonfixing species. All the cyanobacteria capable of fixing nitrogen, contained sym-homospermidine as the major polyamine. The concentration of putrescine, spermidine and spermine was extremely low in these cyanobacteria. The cyanobacteria which normally fail to fix nitrogen contained spermidine as the major polyamine, while the sym-homospermidine content was very low or under the limits of detection. Apparently there is a close relationship between the sym-homospermidine content and the ability to fix nitrogen in cyanobacteria.     

Feeding dynamics of the copepod Diacyclops thomasi before, during and following filamentous cyanobacteria blooms in a large, shallow temperate lake

Cyanobacteria blooms are an increasing problem in temperate freshwater lakes, leading to reduced water quality and in some cases harmful effects from toxic cyanobacteria species. To better understand the role of zooplankton in modulating cyanobacteria blooms, from 2008 to 2010 we measured water quality and plankton abundance, and measured feeding rates and prey selectivity of the copepod Diacyclops thomasi before, during and following summertime cyanobacteria blooms in a shallow, eutrophic lake (Vancouver Lake, Washington, USA). We used a combined field and experimental approach to specifically test the hypothesis that copepod grazing was a significant factor in establishing the timing of cyanobacteria bloom initiation and eventual decline in Vancouver Lake. There was a consistent annual succession of zooplankton taxa, with cyclopoid copepods (D. thomasi) dominant in spring, followed by small cladocerans (Eubosmina sp.). Before each cyanobacteria bloom, large cladocerans (Daphnia retrocurva, Daphnia laevis) peaked in abundance but quickly disappeared, followed by brief increases in rotifers. During the cyanobacteria blooms, D. thomasi was again dominant, with small cladocerans abundant in autumn. Before the cyanobacteria blooms, D. thomasi substantially consumed ciliates and dinoflagellates (up to 100% of prey biomass per day), which likely allowed diatoms to flourish. A shift in copepod grazing toward diatoms before the blooms may have then helped to facilitate the rapid increase in cyanobacteria. Copepod grazing impact was the highest during the cyanobacteria blooms both years, but focused on non-cyanobacteria prey; copepod grazing was minimal as the cyanobacteria blooms waned. We conclude that cyclopoid copepods may have an indirect role (via trophic cascades) in modulating cyanobacteria bloom initiation, but do not directly contribute to cyanobacteria bloom decline.     

Dryland biological soil crust cyanobacteria show unexpected decreases in abundance under long‐term elevated CO2

Biological soil crusts (biocrusts) cover soil surfaces in many drylands globally. The impacts of 10 years of elevated atmospheric CO₂ on the cyanobacteria in biocrusts of an arid shrubland were examined at a large manipulated experiment in Nevada, USA. Cyanobacteria‐specific quantitative PCR surveys of cyanobacteria small‐subunit (SSU) rRNA genes suggested a reduction in biocrust cyanobacterial biomass in the elevated CO₂ treatment relative to the ambient controls. Additionally, SSU rRNA gene libraries and shotgun metagenomes showed reduced representation of cyanobacteria in the total microbial community. Taxonomic composition of the cyanobacteria was similar under ambient and elevated CO₂ conditions, indicating the decline was manifest across multiple cyanobacterial lineages. Recruitment of cyanobacteria sequences from replicate shotgun metagenomes to cyanobacterial genomes representing major biocrust orders also suggested decreased abundance of cyanobacteria sequences across the majority of genomes tested. Functional assignment of cyanobacteria‐related shotgun metagenome sequences indicated that four subsystem categories, three related to oxidative stress, were differentially abundant in relation to the elevated CO₂ treatment. Taken together, these results suggest that elevated CO₂ affected a generalized decrease in cyanobacteria in the biocrusts and may have favoured cyanobacteria with altered gene inventories for coping with oxidative stress.     

Abundance and Diversity of Picocyanobacteria in Shallow Hydrothermal Vents of Panarea Island (Italy)

The abundance and diversity of cyanobacteria living in sediments collected from six vents (temperature range 37–130°C) close to the Panarea Island (Italy) were studied. The diversity of cyanobacteria was analyzed by using a PCR-DGGE approach with group-specific primers. The greatest number and diversity of cyanobacteria were registered at the coldest vent. Sequencing of DGGE fragments revealed that most sequences matched with uncultured cyanobacteria deposited in databases and some were distantly affiliated with different species of Synechococcus. New, genetically distant ribotypes of cyanobacteria are the dominant oxygenic phototrophs in shallow hydrothermal system of Panarea Island.     

蓝藻无菌化方法研究进展

随着微藻产业的蓬勃发展,蓝藻作为具有重要经济价值的资源受到人们越来越多的关注,其多糖和蛋白的开发已被应用于多个领域。为了更深入清晰地挖掘蓝藻的特性和功能,研究者首先需获得无菌的纯种藻株,这是深入开展蓝藻生理生化、遗传和毒性等研究的基础。重点综述了如何根据蓝藻的结构及生理特性选择合适的无菌化方法,介绍了基于蓝藻与污染菌之间差异性的无菌化方法,以期为蓝藻的无菌化工作提供参考。     

Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria

Cyanobacteria are of great importance to Earth's ecology. Due to their capability in photosynthesis and C1 metabolism, they are ideal microbial chassis that can be engineered for direct conversion of carbon dioxide and solar energy into biofuels and biochemicals. Facilitated by the elucidation of the basic biology of the photoautotrophic microbes and rapid advances in synthetic biology, genetic toolkits have been developed to enable implementation of nonnatural functionalities in engineered cyanobacteria. Hence, cyanobacteria are fast becoming an emerging platform in synthetic biology and metabolic engineering. Herein, the progress made in the synthetic biology toolkits for cyanobacteria and their utilization for transforming cyanobacteria into microbial cell factories for sustainable production of biofuels and biochemicals is outlined. Current techniques in heterologous gene expression, strategies in genome editing, and development of programmable regulatory parts and modules for engineering cyanobacteria towards biochemical production are discussed and prospected. As cyanobacteria synthetic biology is still in its infancy, apart from the achievements made, the difficulties and challenges in applying and developing genetic toolkits in cyanobacteria for biochemical production are also evaluated.     

The Cyanobacterial Role in the Resistance of Feather Mosses to Decomposition—Toward a New Hypothesis

Cyanobacteria-plant symbioses play an important role in many ecosystems due to the fixation of atmospheric nitrogen (N) by the cyanobacterial symbiont. The ubiquitous feather moss Pleurozium schreberi (Brid.) Mitt. is colonized by cyanobacteria in boreal systems with low N deposition. Here, cyanobacteria fix substantial amounts of N₂ and represent a potential N source. The feather moss appears to be resistant to decomposition, which could be partly a result of toxins produced by cyanobacteria. To assess how cyanobacteria modulated the toxicity of moss, we measured inhibition of bacterial growth. Moss with varying numbers of cyanobacteria was added to soil bacteria to test the inhibition of their growth using the thymidine incorporation technique. Moss could universally inhibit bacterial growth, but moss toxicity did not increase with N₂ fixation rates (numbers of cyanobacteria). Instead, we see evidence for a negative relationship between moss toxicity to bacteria and N₂ fixation, which could be related to the ecological mechanisms that govern the cyanobacteria – moss relationship. We conclude that cyanobacteria associated with moss do not contribute to the resistance to decomposition of moss, and from our results emerges the question as to what type of relationship the moss and cyanobacteria share.     

Physiological and molecular diversity of feather moss associative N2-fixing cyanobacteria

Cyanobacteria colonizing the feather moss Pleurozium schreberi were isolated from moss samples collected in northern Sweden and subjected to physiological and molecular characterization. Morphological studies of isolated and moss-associated cyanobacteria were carried out by light microscopy. Molecular tools were used for cyanobacteria identification, and a reconstitution experiment of the association between non-associative mosses and cyanobacteria was conducted. The influence of temperature on N2 fixation in the different cyanobacterial isolates and the influence of light and temperature on N2-fixation rates in the moss were studied using the acetylene reduction assay. Two different cyanobacteria were effectively isolated from P. schreberi: Nostoc sp. and Calothrix sp. A third genus, Stigonema sp. was identified by microscopy, but could not be isolated. The Nostoc sp. was found to fix N2 at lower temperatures than Calothrix sp. Nostoc sp. and Stigonema sp. were the predominant cyanobacteria colonizing the moss. The attempt to reconstitute the association between the moss and cyanobacteria was successful. The two isolated genera of cyanobacteria in feather moss samples collected in northern Sweden differ in their temperature optima, which may have important ecological implications.     

Feeding and Refuge Use by Small Fish in the Presence of Cyanobacteria Blooms

We studied the effect of cyanobacteria on foraging and refuge use in small fish. We measured pike larval feeding in the presence of cyanobacteria by counting leftover prey. Our results showed that feeding by pike larvae on zooplankton prey decreased significantly in the presence of non-toxic cyanobacteria. The behaviour can be due to lowered vision caused by turbidity or clogging of the gills. Further, we tested whether the three-spined stickleback use toxic cyanobacteria as a refuge against predators in a choice experiment. The choice experiment was performed in a Y-maze fluviarum, where the fish could select between two different environments. Our results support the refuge use hypothesis because the three-spined stickleback clearly preferred toxic cyanobacteria to the chemical predator signal. To conclude, cyanobacteria decrease feeding rates in fish larvae, but may function as important refuge for e.g. sticklebacks, during predation pressure in pelagic algal blooms.     

水华蓝藻对鱼类的营养毒理学效应

水体富营养化导致蓝藻水华的发生已成为全球关注的水环境问题,很多鱼类处于水生态系统食物链的最高级,蓝藻水华的主要次级代谢产物-微囊藻毒素可通过鱼类的摄食活动或生物富集作用在鱼体组织中累积,并通过食物链危及人类健康。近年来,微囊藻毒素对鱼类的毒性效应引起众多科学家的关注。在天然水体中不少鱼类可以主动摄食蓝藻,所以,水华蓝藻对鱼类来说既具有营养物作用、也具有潜在的毒性作用。鉴于目前机械收获的水华蓝藻生物量资源化利用问题以及水产饲料业亟需大力开发鱼粉替代蛋白源的需要,从营养学和毒理学这两个角度来研究水华蓝藻对鱼类的营养作用和毒性效应具有较高的理论和现实意义。主要概述了蓝藻粉、蓝藻细胞对鱼类的营养学和毒理学效应,以期拓展水华蓝藻对鱼类毒性效应的研究视野,同时也为水华蓝藻的资源化利用提供新的思路。     

启动子Ptac与PsbA在鱼腥藻7120中表达hG-CSF的效率比较

为了比较外源性启动子Ptac与内源性启动子PsbA在鱼腥藻7120中表达外源基因时的效率,构建了分别含Ptac和PsbA两种启动子的穿梭表达载体pRL-PsbA-GCSF、pRL-Tac-GCSF;利用三亲结合转移法转化鱼腥藻7120,利用抗生素筛选,通过质粒提取和PCR方法鉴定,获得了分别由2种启动子驱动表达hG-CSF的转基因蓝藻,转基因藻中目的基因以质粒形式存在;利用半定量RT-PCR方法对2种转基因藻的hG-CSF转录水平进行比较,发现PsbA启动子驱动效率与Ptac启动子没有明显差异;利用ELISA方法比较hG-CSF蛋白表达量,发现PsbA启动的蓝藻中hG-CSF表达量是Ptac诱导条件下表达量的1.17倍。     

Allelopathy among Cyanoprokaryota and Other Algae Originating from Lake Neusiedlersee (Austria)

During the late eighties, the eutrophic shallow lake Neusiedlersee located at the austrian – hungarian border showed a massive development of cyanobacteria, which coincided with a strong decline of chlorococcales. Besides a change in nutrient conditions, allelopathy may have contributed to the dominance of cyanobacteria, too. To get a first insight into potential allelopathic effects, 13 cyanobacteria strains were tested originating for the most part from lake Neusiedlersee. Plate diffusion assays showed allelopathic activity of 6 strains indicating chemical interactions between cyanobacteria and other algae.